To date, the only vaccine trial (RV144) that has shown any protective efficacy against HIV acquisition is based on a poxvirus prime?protein boost immunization strategy. Although the efficacy achieved was modest (~31%), these findings provide a strong rationale to seek improvements for the prime-boost immunization approach and to gain better insight on the nature of the protective immunity achieved. In this application, we seek to improve responses to prime boost immunization by a combination of three independent approaches: (i) to present stabilized trimeric Env spikes on virus-like particles (VLP) as immunogens for vaccinia virus prime and VLP boost; (ii) to incorporate stabilized trimeric envelope (Env) from multiple isolates as polyvalent VLP immunogen to increase the breadth of response; and (iii) to explore approaches that may increase the number of SOSIP trimer spikes on VLP. Our overall working hypothesis is that the protective efficacy of prime-boost immunization can be improved by optimizing immunogen and immunization regimen to enhance the breadth and potency of both neutralizing and non-neutralizing antibody responses that have been associated with protection in human and/or non-human primate models. Specifically, we hypothesize that by presenting stabilized trimeric Env on VLP in the poxvirus-protein prime boost regimen, we will be able to enhance neutralizing antibody responses, and by using polyvalent Env and increasing the density of Env spikes on the VLP immunogens, we will further amplify the breadth and the potency of response. The enhanced breadth and potency of both neutralizing and non-neutralizing antibody responses, including V1/V2-directed antibodies and those that mediate antiviral effector functions, such as ADCC, will contribute to the protective efficacy of the poxvirus-protein prime boost immunization platform. The Specific Aims of this proposal are: (1) To determine the structure and the antigenic and immunogenic profiles of stabilized Env trimers incorporated into VLP; (2) To determine if stabilized Env trimers from multiple isolates can be incorporated on VLP and if such polyvalent vaccines when used in a prime/boost regimen will increase the breadth of Nab and non-Nab responses; (3) To determine if cytoplasmic tail modifications will increase the density of stabilized Env spikes on VLP and if increased Env density will enhance the breadth and potency of Env specific responses; and (4) To examine if immunization regimens down-selected from the preceding studies in rabbits can be translated to macaques. If successful, insights obtained from these studies will inform the clinical development of vaccines and vaccine strategies that may be more effective than those used in RV144 to prevent HIV-1 acquisition in humans.